| Drug addiction,as a chronic brain disease with complicated pathological mechanism and high liability to relapse,involves a series of advanced neuropsychiatric dysfunction including reward,learning memory,motivation,decision-making,execution and central control,has become a serious health and social problem.According to the China Drug Report 2018,there are 2.404 million drug addicts nationwide,among which 1.35 million are methamphetamine addicts,accounting for 56.1%,suggesting that methamphetamine has become the most popular substance of abuse.Compared with classic drugs,methamphetamine has higher potentials for physical and psychological dependence,thus harms the central nervous system seriously and causes irreversible damages in addicts.In addition,methamphetamine addition results in serious problems of society,economy and public health.However,till now,the neurobiological mechanism underlying methamphetamine addiction remains unclear,and neither effective intervention nor therapeutic measures is availableAccumulating research suggests that the key to treat drug addiction is to solve the problem of relapse,in which eliminating the "intractable" addiction memory is vital.Drug addiction memory is a long-lasting pathological learning memory which formed under the comprehensive influence of positive reward,withdrawal-induced negative emotions and drug-related clues.Drug addiction memory is reluctant to fade,and relapse can occur even after abstinence for several years or a decade.In clinical,many factors can induce relapse,including drug-related cues,priming drug and stress.Numerous studies demonstrate that drug-related cues,as one of the most important factors that induce craving in clinical,play an extremely important role in the formation,maintenance,and relapse of drug addiction.Although the functions of drug-related cues can be attributed to their conditioned reinforcing and incentive motivational properties,the mechanism underlying methamphetamine addiction and relapse remains unclear.Associative learning and memory are common ways for information acquisition,it is essential for logical reasoning,associative thinking,comparison,and computation.Associated learning refers to that when two or more events have repeatedly spatiotemporal interactions,they can have mutual correlation,so that the individual learns that the occurrence of one event means the occurrence of others.There are two types of associated learning-Pavlovian conditioning and instrumental conditioning.It has been reported by Professor Jinhui Wang that after Pavlovian associative learning and memory of a whisker stimulus and an olfactory stimulus,the whisker cortical neurons can be activated by the olfactory stimulus,resulting in the whisker stimulus-related behavior,vice versa,suggesting that the effects of associative learning and memory on both stimulus are bidirectional.Furthermore,the establishment of associative learning and memory were accompanied by the increase projections between related sensory cortex and structural and functional synaptic changes in related sensory cortex,suggesting that the associative memory cells might be the material basis of the associative learning and memory among sensory stimulus.Considering that drug addiction is also a pathological learning and memory associated with discrete,discriminative,and contextual cues,is it possible that the associative learning and memory mechanism also plays a role in methamphetamine addiction?Using the Pavlovian-to-Instrumental Transfer(PIT)model,elegant prior studies have separated the Pavlovian conditioning of cues and addictive drugs from the instrumental response,and implicate that it is the incentive motivation but not the conditioned reinforcement of drug-related cues that results in relapse.However,further research demonstrated that cue extinction therapy conducted outside the drug-taking context,while decreased subjective cravings and physiological responses to drug-related cues,was not effective to reduce relapse,suggesting that there must be other mechanisms underlying cue-induced relapse other than incentive motivation.Given that drug-induced relapse is normally attributed to interoceptive awareness with the unknown mechanism to date,is it possible that the bidirectional effects of associative learning and memory on both methamphetamine and drug-related cues alter the process of methamphetamine addiction as well as cue-or drug-induced relapse?Previous studies on drug addiction only focused on the influence of associative learning on drug-related cues,but alterations of drug’s effects have been overlooked for a long time.Our scientific hypothesis holds that addiction memory is likely to be similar to sensory associative memory.The pathological associative learning and memory can be formed among methamphetamine-related cues and the rewarding effects of methamphetamine,and stored in the associative memory cells.Thus,methamphetamine-related cues cause methamphetamine-related response,while the presence of methamphetamine trigger response to methamphetamine-related cues.In this process,associated learning enhances the previous connection between the methamphetamine-and cues-related brain regions,and forms new projections and synaptic connections,thus promotes the formation and maintenance of methamphetamine addiction through facilitating the learning process.Meanwhile,both methamphetamine and cues can activate the associative memory cells,which store both drug and cue-related memories,thereby induces drug-taking and drug-seeking behaviors.As a result,not only methamphetamine-related cues can induce the craving and relapse in the absence of methamphetamine,the response induced by methamphetamine itself may also be enhanced.In order to prove this hypothesis,we must find evidence at the level of behavior and neurobiology.This study is theoretically important to elucidate the mechanism underlying methamphetamine addiction,and may also provide new clues and new ideas for interventions and therapeutics of methamphetamine addictionExperimental methods1.Using the classic and the modified self-administration models in mice,to investigate the effects of the associative learning and memory between the visual cue and methamphetamine,accompanying the instrumental behaviors,on drug-taking behaviors induced by both methamphetamine+visual cue and methamphetamine alone during the acquisition2.Using the modified PIT model in mice,further research was performed to differ the effects of Pavlovian conditioning between methamphetamine and the visual cue from conditioned reinforcing effects of the visual cue on the drug-taking behaviors induced by methamphetamine alone during the acquisition and contextual extinction without methamphetamine or the visual cue.3.To verify the manipulation of virus system,and to lay the foundation to label primary associative cells in the drug-stimulated primary brain region ventral tegmental area(VTA)and the visual cue-stimulated primary brain regions striatum cortex V1(SCV1),and to reveal the associated learning and memory mechanism underlying methamphetamine addictionResults1.The effects of the association between visual cue and methamphetamine delivery accompanied the instrumental response on methamphetamine addiction behavior(1)During the formation,the association of the visual cue and methamphetamine delivery accompanied the instrumental response facilitated the acquisition of methamphetamine-induced self-administration and enhanced methamphetamine alone-induced drug-taking in miceDuring the formation period of the classic self-administration model,using the fixed ratio 1(FR1,touch an active nosepoke can receive one infusion of drug)training procedure,during the 11 days training period(formation period,3 hours/day,the maximum number of infusions is 100),mice were randomly divided into Paired Stimulation Group(PS group,one methamphetamine infusion accompany with 5 s active poke light),Unpaired Stimulation Group(UPS group,random 5 s active poke light was not associated with methamphetamine infusion)and Control Group(Con group,no active poke light).The results showed that compared with UPS group and Con group,the number of methamphetamine(0.05 mg/kg/infusion)infusions in PS group were significantly increased(Two-way RM ANOVA:cue pairing main effect P<0.01,time main effect P<0.001,interaction P<0.001,n=6).Drug alone test was conducted on the Day 12,all mice in the three groups touch an active nosepoke can receive one infusion of methamphetamine(0.05 mg/kg/infusion),no visual cue(no active nosepoke light).The results showed that compared with UPS group and Con group,the number of methamphetamine(0.05 mg/kg/infusion)infusions in PS group were significantly increased(one-way ANOVA:cue pairing main effect P<0.05,n=6)The above results suggest that during the formation,the association of the visual cue and methamphetamine delivery accompanied the instrumental response facilitated the acquisition of methamphetamine-induced self-administration and enhanced methamphetamine alone-induced drug-taking in mice.However,in this model,the total methamphetamine intake in PS group was significantly higher than that in UPS group and Con group(one-way ANOVA:cue pairing main effect P<0.05,n=6),it is suggested that the total methamphetamine intake of three groups were unequal,the more methamphetamine of the PS group may result in higher reinforcing effects of methamphetamine in the PS group.Therefore,it is impossible to distinguish whether the enhancement of drug self-effect here is the direct effect of associative learning and memory or the indirect effect of associative learning and memory through enhancing instrumental behavior,increasing methamphetamine intake and reward effect in PS group.(2)Under the same methamphetamine intake,the association of the visual cue and methamphetamine delivery accompanied the instrumental response directly increased the motivation of drug-taking during the acquisition and showed bidirectional enhancement of responses to both methamphetamine alone and cue alone in miceTo investigate whether the associative learning and memory have a directly effect on the acquisition of methamphetamine-induced self--administration and the effect of methamphetamine itself,we must rule out the impact of different rewarding effects induced by unequal methamphetamine intake of three groups.Thus,we modified the training mode of the classic self-administration model.The modified self-administration model(training mode 2)adopts FR1 training procedure.The mice were still divided into PS group(one methamphetamine infusion accompany with 5 s active nosepoke light),UPS group(random 5 s active nosepoke light was not associated with methamphetamine inf-usion)and Con group(no active nosepoke light).The time spent in obtaining 30 methamphetamine infusions(0.05 mg/kg/time)of each group in each session is measured to evaluate the motivation of mice.During the 12 days self-administration formation phase,compared with UPS group and Con group,the time spent in obtaining 30 methamphetamine infusions in PS group were significantly reduced(Two-way RM ANOVA:cue pairing main effect P<0.001,time main effect P>0.05,interaction P>0.05,n=8～9).Drug alone test was conducted on the Day 13,all mice in the three groups touch an active nosepoke can receive one infusion of methamphetamine(0.05 mg/kg/infusion),no visual cue(no active nosepoke light).The results showed that compared with UPS group and Con group,the time spent in obtaining 30 methamphetamine infusions in PS group were significantly reduced(one-way ANOVA:cue pairing main effect P<0.001,n=8～9).Cue alone test was conducted on the Day 14,all mice in the PS group and UPS group touch an active nosepoke can receive 5 s active nosepoke light,no infusion of methamphetamine.The results showed that compared with UPS group,the time spent in obtaining 30 cues in PS group were significantly reduced(one-way ANOVA:cue pairing main effect P<0.01,n=9).These results suggested that the association of the visual cue and methamphetamine delivery accompanying the instrumental response directly increased the motivation of drug-taking induced by methamphetamine+visual cue or methamphetamine alone,and the motivation of drug-seeking induced by the visual cue alone during the acquisition.Thus,the associative learning and memory not only directly increased the motivation of drug-taking but also showed bidirectional enhancement of responses to both methamphetamine alone and cue alone during the acquisition.2.The effects of Pavlovian association of the visual cue and passive methamphetamine delivery on methamphetamine addiction behaviorPrevious studies attributed the drug-related cue-induced enhancement of drug addiction and induced relapse after extinction to conditioned reinforcing effects and incentive motivation required by cues.However,our findings demonstrated that after establishment of associative learning and memory,the drug-taking behaviors induced by methamphetamine increased even with absent of cues,suggesting other mechanism involved in drug-related cues-induced enhancing acquisition of addiction.In order to investigate whether the direct action of Pavlovian learning or the indirect action induced by the conditioned reinforcement of cues was account for the increased effects of methamphetamine itself,the modified PIT model was used to separated Pavlovian training sessions with instrumental sessions.(1)The Pavlovian association of the visual cue and passive methamphetamine delivery enhanced the acquisition of methamphetamine-induced self-administration directly by increasing the motivation of drug-taking induced by methamphetamine alone in miceIn the modified PIT model,the 8 days FR1 training phase(3 hours/day,the maximum number of infusions is 100,no visual cue)was use to established the stable methamphetamine(0.05 mg/kg/infusion)-induced self-administration.Pre-test was conducted on the Day 9,all mice touch an active nosepoke can receive one infusion of methamphetamine(0.05 mg/kg/infusion),no visual cue(no active nosepoke light),the average number of methamphetamine infusions per mouse on the Day 6-8 was set as the task quantity to each mouse,and the time spent on each mouse to completing the task quantity was measured.After pre-test,according to their task quantity,the time spent to completing the task quantity in the pretest,methamphetamine intake of Day 1-9,the mice were equally divided into paired cue stimulation group(PS group),unpaired cue stimulation group(UPS group)and control group(Con group).The Pavlovian association of the visual cue and methamphetamine were established on the Day 10 and Day 14,for 3 hours/day.In this stage,mice receive methamphetamine(0.05 mg/kg/infusion)infusion passively,and the number of infusions receive by each mouse was equal to the task quantity of pre-test,so as to ensure that the reward intensity received by each animal would not change suddenly.PS group receive methamphetamine infusion accompany with 5 s active nosepoke light passively,5 s active nosepoke light of the UPS group random show and not associated with methamphetamine passively infusion,Con group just passively infusion with methamphetamine,no active nosepoke light.Post-test was conducted on the Day 11-1311th to 13th and Day 15-16,that was drug alone test,no visual cue(no active nosepoke light),and the method was exactly the same as day 9.The results showed that compared with UPS group and Con group,the time spent in completing the task quantity of PS group were significantly reduced(Tow-way ANOVA:cue pairing main effect P<0.001,time main effect P>0.05,interaction P>0.05,n=4).The result suggesting the Pavlovian association of the visual cue and passive methamphetamine delivery enhanced the acquisition of methamphetamine-induced self-administration directly by increasing the motivation of drug-taking induced by methamphetamine alone in mice.These results revealed that P avlovian associative learning and memory not only the basis of conditioned reinforcing effects and incentive motivation of drug-related cues,but also has a directly effect of contributed to the acquisition of methamphetamine addition.This is behavioral evidence of the existence of methamphetamine addiction associative learning and memory.(2)The Pavlovian association of the visual cue and passive methamphetamine delivery attenuated the environmental extinction in miceTo further investigate the influence of associative learning and memory on methamphetamine extinction and relapse,we performed 14 days extinction sessions since Day 17.During the extinction period,all mouse in three groups touch active nosepoke or inactive nosepoke was only recorded,neither methamphetamine delivery nor the visual cue(no active nosepoke light).The results show that compared with UPS group and Con group,the number of active pokes in PS group during contextual extinction were significantly increased(Tow-way ANOVA:cue pairing main effect P<0.05,time main effect P<0.001,interaction P>0.05,n=4),suggesting that Pavlovian association of the visual cue and passive methamphetamine delivery attenuated the extinction induced by environment.The above results show that the Pavlovian association of the visual cue and passive methamphetamine delivery in the formative period made methamphetamine addiction more difficult to fade.Above all,in the process of establishing the Pavlovian association of visual cues-methamphetamine in PS group,the effects of methamphetamine and visual cues themselves are enhanced,and both of them establish stronger association learning and memory with environmental cues,so that enabling environmental cues to activate more memory cells and induce stronger motivation to seek drugs when extinction.This is the second behavioral evidence of the existence of methamphetamine addiction-related associative memory.3.Validation of Tet on conditional gene expressions system for labeling methamphetamine addiction-related associative memory cellsThrough the research in the first two chapters,it is clear from the behavior that associative learning and memory participate in the formation of methamphetamine addiction behavior,is the reason why addiction behavior is difficult to fade.In order to clarify the neurobiological mechanism underlying associative learning and memory participating in methamphetamine addiction,we need to find the material basis of the associative learning and memory mechanism of addiction,that is the methamphetamine addiction-related associative memory cells.In the training mode 2,Tet on conditional gene expression system will be used to mark nerve cells only activated by methamphetamine(called one-phase marker)in methamphetamine alone test,and nerve cells only activated by visual cues(called two-phase marker)will be marked by Fos immunofluorescence staining in visual cue alone test.When a nerve cell is labeled by Tet on system and Fos simultaneously,we preliminarily judge it to be an addictive associative learning and memory cell that stores methamphetamine and visual cue related memory.Tet on system belongs to tetracycline regulation system.Its spatio-temporal specific labeling of cells depends on the expression of Fos in brain and exogenous doxycycline(Dox).In order to determine the feasibility of Tet on system in methamphetamine addiction associative learning memory cell labeling and the optimal induction time of Dox.We injected Tet on virus system into mouse VTA,and gave Dox at 3 time points of 6 days,3 days and 1 day before methamphetamine stimulation.We verified Tet on system by observing the expression of mCherry and Fos in VTA.After 2 weeks of virus expression,mice were stimulated with methamphetamine(1 mg/kg,i.p.)or normal saline(i.p.),after 4 hours the brains were taken,and prepare for immunofluorescence experiment.The immunofluorescence results showed that,after only one day administration of Dox,the Tet on system was able to switch on mCherry expression depending on methamphetamine activation,while no mCherry expression was detected without Dox,indicating that the switch of the Tet on system is regulated by Dox.In the presence of Dox,compared with the saline group,cells in VTA expressing both Fos and mCherry increased after methamphetamine stimulation,indicating that the methamphetamine can switch on the Tet on system by increasing the Fos expression in VTA.And compared with the Dox group given 6 days before stimulation and 3 days before stimulation,the nonspecific expression of mCherry(expression caused by non-methamphetamine stimulation)after intraperitoneal saline injection was lower in the Dox group given 1 day before stimulation.The above results show that methamphetamine can induce the activation of Tet on system by activating the expression of Fos in the presence of Dox,and administration of Dox one day before stimulation can reduce the nonspecific expression of mCherryConclusionIn summary,this study revealed that like associative learning of multiple sensory stimuli,the effects of the associative learning and memory on enhancing responses to both methamphetamine and cue were bidirectional.Furthermore,it was the Pavlovian associative learning and memory that directly enhanced the acquisition of methamphetamine-induced self-administration by increasing the motivation of drug-taking induced by methamphetamine alone but attenuated the contextual extinction in mice.These results suggest that not only conditioned reinforcing effects and incentive motivation of drug-related cues,but also direct and bidirectional effects of associative learning are account for enhanced methamphetamine addiction and resistance to extinction and highlight the possible associative learning and memory mechanism underlying methamphetamine addiction and relapse,which might be important in mediating the acquisition of methamphetamine addiction and contextual-,discrete cue-,and drug-induced relapse Furthermore,this study also provides a reliable behavioral paradigm for the study of drug addiction-related associative learning and memory,and lays solid foundations for the further labeling associative memory cells and elucidating of the associative learning and memory mechanism underlying methamphetamine addiction.It is promising to reveal the neurobiological mechanism underlying intractable methamphetamine addiction,which is of great significance for the discovery of effective anti-relapse interventions for the treatment of methamphetamine addiction. |
PDF Full Text Request